PubMed Health⌕ Search

Biomedical subjects

K Poralla

Publications and source records attributed to K Poralla.

At least 19 recordsLinked to original sources
PubMed2000

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.

Culture Media↗
PubMed2000

Conserved tyr residues determine functions of Alicyclobacillus acidocaldarius squalene-hopene cyclase.

The catalytic cavity of Alicyclobacillus acidocaldarius squalene-hopene cyclase is mainly lined by aromatic amino acids. In recombinant cyclases, three out of four tyrosine residues (Y) have been mutated to phenylalanine residues (F). The mutant cyclases Y495F and Y612F had less activity than the wild-type cyclase, but a wild-type product pattern. Mutant Y609F had wild-type activity but a drastically altered product pattern with hopene and significant amounts of bicyclic alpha-polypodatetraene and different tetracyclic triterpenes (dammaradienes and eupha-7,24-diene). The experiments demonstrated that Y495 and Y612 may be involved in the initiation of the cyclization reaction and Y609 in the stabilization and/or positioning of the intermediate carbocations.

Bacillus↗
PubMed2000

Rationally designed inhibitors as tools for comparing the mechanism of squalene-hopene cyclase with oxidosqualene cyclase.

The inhibition of squalene-hopene cyclase (SHC) (E.C. 5.4.99.-), an enzyme of bacterial membranes catalyzing the formation of pentacyclic sterol-like triterpenes, was studied by using different classes of compounds originally developed as inhibitors of oxidosqualene cyclase (OSC) (E.C. 5.4.99.7), the enzyme of eukaryotes responsible for the formation of tetracyclic precursors of sterols. The mechanism of cyclization of squalene by SHC, beginning with a protonation of the 2,3 double bond by an acidic residue of the enzyme, followed by a series of electrophilic additions of the carbocationic intermediates to the double bonds, is similar to the mechanism of cyclization of 2,3-oxidosqualene by OSC. The inhibitors studied included: (i) analogs of the carbocationic intermediates formed during cyclization, such as aza-analogs of squalene and 2,3-oxidosqualene; (ii) affinity-labeling inhibitors bearing a methylidene reactive group; and (iii) vinyldioxidosqualenes and vinylsulfide derivatives of the substrates. Comparison of the results obtained with the two enzymes, SHC and OSC, showed that many of the most effective inhibitors of OSC were also able to inhibit SHC, while some derivatives acted as specific inhibitors. Differences could be easily explained on the basis of the different substrate specificity of the two enzymes.

Animals↗
PubMed1998

Cloning of conserved genes from Zymomonas mobilis and Bradyrhizobium japonicum that function in the biosynthesis of hopanoid lipids.

The squalene-hopene cyclase (SHC) is the only enzyme involved in the biosynthesis of hopanoid lipids that has been characterized on the genetic level. To investigate if additional genes involved in hopanoid biosynthesis are clustered with the shc gene, we cloned and analyzed the nucleotide sequences located immediately upstream of the shc genes from Zymomonas mobilis and Bradyrhizobium japonicum. In Z. mobilis, five open reading frames (ORFs, designated as hpnA-E) were detected in a close arrangement with the shc gene. In B. japonicum, three similarly arranged ORFs (corresponding to hpnC-E from Z. mobilis) were found. The deduced amino acid sequences of hpnC-E showed significant similarity (58-62%) in both bacteria. Similarities to enzymes of other terpenoid biosynthesis pathways (carotenoid and steroid biosynthesis) suggest that these ORFs encode proteins involved in the biosynthesis of hopanoids and their intermediates. Expression of hpnC to hpnE from Z. mobilis as well as expression of hpnC from B. japonicum in Escherichia coli led to the formation of the hopanoid precursor squalene. This indicates that hpnC encodes a squalene synthase. The two additional ORFs (hpnA and hpnB) in Z. mobilis showed similarities to enzymes involved in the transfer and modification of sugars, indicating that they may code for enzymes involved in the biosynthesis of the complex, sugar-containing side chains of hopanoids.

Amino Acid Sequence↗
PubMed1998

Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids.

We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene-hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS.

Amino Acid Sequence↗
PubMed1997

Structure and function of a squalene cyclase.

The crystal structure of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was determined at 2.9 angstrom resolution. The mechanism and sequence of this cyclase are closely related to those of 2,3-oxidosqualene cyclases that catalyze the cyclization step in cholesterol biosynthesis. The structure reveals a membrane protein with membrane-binding characteristics similar to those of prostaglandin-H2 synthase, the only other reported protein of this type. The active site of the enzyme is located in a large central cavity that is of suitable size to bind squalene in its required conformation and that is lined by aromatic residues. The structure supports a mechanism in which the acid starting the reaction by protonating a carbon-carbon double bond is an aspartate that is coupled to a histidine. Numerous surface alpha helices are connected by characteristic QW-motifs (Q is glutamine and W is tryptophan) that tighten the protein structure, possibly for absorbing the reaction energy without structural damage.

Amino Acid Sequence↗
PubMed1997

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.

Bacillaceae↗
PubMed1996

Site-directed mutagenesis of putative active-site residues in squalene-hopene cyclase.

Squalene-hopene cyclase (SHC) catalyzes the complex polycylization of squalene to hopene, similar to the cyclization of oxidosqualene to sterols. Sequence analysis of SHC revealed a highly conserved aspartate-rich motif (DDTA), comparable to the DCTA motif of oxidosqualene cyclases, which is supposed to be part of the active site. In order to determine the importance of the motif in squalene cyclization, the conserved residues Asp376 and Asp377 in the DDTA motif of SHC from Alicyclobacillus acidocaldarius were individually replaced by glutamate, glutamine, glycine, and arginine. With the exception of the [Glu376]SHC mutant, all other substitutions resulted in almost or complete loss of enzyme activity. Compared to that of the wild-type enzyme, the specific activity of the [Glu376]SHC mutant enzyme was reduced to 10%, accompanied by a significant decrease in the apparent Vmax, whereas the apparent K(m) remained unchanged, CD measurements indicated that mutations did not affect the secondary structure. It is proposed that Asp376 and Asp377 are crucial for catalysis and may act as point charges to stabilize intermediate cations. Moreover, for squalene-hopene cyclase, a high content of alpha-helical conformation could be found, providing the first structural information for a triterpene cyclase.

Binding Sites↗
PubMed1995

Three new omega-cycloheptyl fatty acids from Alicyclobacillus cycloheptanicus and their biosynthetic interrelationships.

Three minor omega-cycloheptyl fatty acids from Alicyclobacillus cycloheptanicus have been identified as omega-cycloheptylnonanoate, -decanoate, and -alpha-hydroxytridecanoate. The biosynthetic interrelations of these cyclic fatty acids have been studied. 13C-Labeled omega-cycloheptylundecanoate and -alpha-hydroxyundecanoate were converted to omega-cycloheptyldecanoate, which is one carbon shorter in length. alpha-Hydroxylation of omega-cycloheptylundecanate was also observed, but not chain extension to omega-cycloheptyltridecanoate.

Bacillus↗
PubMed1995

Zymomonas mobilis squalene-hopene cyclase gene (shc): cloning, DNA sequence analysis, and expression in Escherichia coli.

Using a DNA probe from the gene encoding squalene-hopene cyclase (SHC, EC 5.4.99.-) from the Gram-positive bacterium Alicyclobacillus acidocaldarius, we have cloned a 4.3 kb HindIII fragment of chromosomal DNA from Zymomonas mobilis. An open reading frame of 1977 bp was detected that could encode a protein of 658 amino acids with a calculated molecular mass of 74077 Da. Under the control of lac or tac promoters, this gene, shc, was expressed in Escherichia coli K12 strains and its product had squalene-hopene cyclase activity. Sequence alignments with the A. acidocaldarius SHC, the lanosterol cyclase of the yeast Candida albicans, and the cycloartenol synthase of the plant Arabidopsis thaliana revealed six highly conserved regions (mainly in the C-terminal part) of the proteins. These regions contained the core motif Gln-X-X-X-Gly-X-Trp.

Amino Acid Sequence↗
PubMed1994

Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol.

The squalene-hopene cyclase of the hopanoid- and tetrahymanol-producing Rhodopseudomonas palustris was released from the isolated membranes by CHAPS and purified to homogeneity by successive chromatography on DEAE Sephacel, Octyl Sepharose, and Blue Sepharose. The enzyme has a molecular weight of 70 kDa as determined by SDS-PAGE and an isoelectric point at about pH 5.0. The enzyme activity has a maximum at 30 degrees C and at pH 6.5. No production of tetrahymanol could be demonstrated by using either crude or purified cyclase preparations.

Amino Acid Sequence↗
PubMed1994

Genetic and biochemical analyses of the biosynthesis of the yellow carotenoid 4,4'-diaponeurosporene of Staphylococcus aureus.

The major pigment produced by Staphylococcus aureus Newman is the deep-yellow carotenoid 4,4'-diaponeurosporene; after prolonged cultivation, this pigment is in part converted to the orange end product staphyloxanthin. From this strain a 3.5-kb DNA fragment was identified which after being cloned into Escherichia coli and Staphylococcus carnosus conferred the ability to produce 4,4'-diaponeurosporene. DNA sequencing of this fragment revealed two open reading frames (ORFs) which are very likely cotranscribed. ORF1 encodes a 254-amino-acid hydrophobic protein, CrtM (M(r), 30,121). The deduced sequence of CrtM exhibits in three domains similarities to the sequences of Saccharomyces cerevisiae and human squalene synthases and phytoene synthases of various bacteria. ORF2 encodes a 448-amino-acid hydrophobic protein, CrtN, with an M(r) of 50,853 whose deduced sequence is similar to those of phytoene desaturases of other bacteria. At the N terminus of CrtN a classical FAD-, NAD(P)-binding domain is found. Spectrophotometry and gas chromatography-mass spectrometry analyses of the carotenoid production of E. coli and S. carnosus clones containing either ORF1 or both ORFs together suggest that ORF1 and ORF2 represent the dehydrosqualene synthase gene (crtM) and the dehydrosqualene desaturase gene (crtN), respectively. The results furthermore suggest that the biosynthesis of 4,4'-diaponeurosporene starts with the condensation of two molecules of farnesyl diphosphate by dehydrosqualene synthase (CrtM); it is shown that the reaction product of this enzyme is dehydrosqualene and not squalene. Dehydrosqualene (4,4'-diapophytoene) is successively dehydrogenated by a desaturase (CrtN) to form the yellow main intermediate 4,4'-diaponeurosporene.

Amino Acid Sequence↗
PubMed1992

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov.

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

Bacillaceae↗
PubMed1992

Structural studies of an emulsion-stabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain.

The primary structure of an emulsion-stabilizing exopolysaccharide from the adhesive, hydrophobic Rhodococcus strain No. 33 was elucidated by NMR spectroscopy, methylation analyses, periodate oxidation and oligosaccharide analyses. The polysaccharide PS-33 consisted of rhamnose, galactose, glucose and glucuronic acid in molar ratios of 2:1:1:1. The main chain contained 3-substituted alpha-D-glucuronic acid linked to the 3-position at alpha-L-rhamnose, in addition to 3-substituted residues of beta-D-galactose and alpha-D-glucose. The alpha-L-rhamnose of the side chain was linked to position 4 of the galactose. In addition, the polysaccharide was O-acetylated, corresponding to one acetyl group per repeating unit. From the results two structural possibilities could be suggested. As the polysaccharide carries hydrophobic groups (methyl of rhamnose/O-acetyl), it is very likely that these are of general significance for the emulsifying activity of polysaccharides. It also seems to be possible that this polysaccharide is at least partially responsible for the hydrophobic cell surface properties of the Rhodococcus strain No. 33 and it may be involved in hydrophobic interactions when adhering to hydrophobic interfaces.

Bacterial Adhesion↗
PubMed1992

Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism.

The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found.

Amino Acid Sequence↗
PubMed1991

Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila.

The membrane-bound enzyme from Tetrahymena thermophila responsible for the conversion of squalene into the quasi-hopanoid tetrahymanol was purified 297-fold to near homogeneity. Purification involved solubilization by octylthioglucoside, chromatography on DEAE-trisacryl, hydroxyapatite and FPLC ion-exchange on Mono Q. The apparent KM was found to be 18 microM. 2,3-Iminosqualene and N,N-dimethyldodecylamine-N-oxide are effective inhibitors of the cyclase with I50 values of 50 and 30 nM, respectively. The cyclase has a molecular mass of 72 kDa as judged by electrophoresis in polyacrylamide gels under denaturating conditions. The optimal enzymatic activity was obtained at pH 7.0 and 30 degrees C. The solubilized enzyme needs the presence of detergent for maintaining activity. The influence of different detergents on cyclase activity was studied. Triton X-100 proved to be a strong inactivator of the enzyme. Solubilization of the cyclase in Tween 80 and digitonin inactivates the enzyme. However, its activity can be recovered by complementation of the assay buffer with octylthioglucoside above its critical micellar concentration. We suggest that this approach might be applicable to other membrane-bound proteins.

Animals↗
PubMed1990

Properties of purified squalene-hopene cyclase from Bacillus acidocaldarius.

The squalene-hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X-100, chromatography on DEAE-cellulose, phenyl Sepharose and two gel-filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n-alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate, dodecyldimethylamine N-oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate and dodecyldimethylamine N-oxide was demonstrated by Lineweaver-Burk plots.

Amino Acid Sequence↗